Pharmaceutical composition for promoting angiogenesis

ABSTRACT

The present invention provides a pharmaceutical composition for promoting angiogenesis, which has an angiogenesis promoting action even in a vascular culturing system without effect of microcirculation. A pharmaceutical composition for promoting angiogenesis of the present invention comprises at least one compound selected from a group consisting of a piperidine compound represented by the general formula (1) or salts thereof: (wherein R represents a benzoyl group which may have, as substituents on the phenyl ring, 1 to 3 groups selected from a group consisting of an amino group, which may have a lower alkanoyl group, and a lower alkyl group; R 1  represents a hydrogen atom or a lower alkyl group; and R 2  represents a phenyl lower alkyl group).

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition forpromoting angiogenesis.

BACKGROUND ART

Angiogenesis is a process for formation of a systemic vascular networkinitiating from embryonic stage, and is related to generation viacomplex processes including not only proliferation of vascularendothelial cells but also migration of endothelial cells or tubeformation, formation of basement membrane, etc.

Recently, studies on factors regulating angiogenesis have beenprogressed, and therapeutic application thereof has been attempted.Development of tumors and the like profoundly relates to dysregulatedangiogenesis. Treatment thereof requires suppression of angiogenesis,however, on the contrary, therapy for promoting angiogenesis has alsobeen required widely.

For example, factors for promoting angiogenesis, vascular endothelialgrowth factor (VEGF), basic fibroblast growth factor (bFBF), hepatocytegrowth factor (HGF), and the like have been found, and by utilizingthese growth factors and genes thereof, therapeutic methods for thediseases essentially requiring improvement of blood circulation, such asarteriosclerosis obliterans, ischemic heart disease, and the like, havebeen attempted and examined.

However, since these growth factors are proteins, there are manyproblems such as difficulty with the oral administration, ananaphylactic reaction caused by repeated administration, safety of virusin case of gene therapy using a viral vector and adverse side reactionslike edematous, and accordingly new development of therapeutic agentsare desired.

Physiological angiogenesis is recognized in microvascular formation ofmature tissues and is known to be generated by such a physical factor aschange in intravascular pressure. It has been reported, for example,that long-term administration of prazosin or adenosine havingvasodilating activity increases physical shear stress to blood vesselsin microcirculation and thus promotes angiogenesis (Dawson, J. M.,Cardiovasc. Res. 23, 913-920, 1989; and Ziada, A. M., Cardiovasc. Res.18, 724-732, 1984), however, these compounds have no direct angiogenesispromoting action.

DISCLOSURE OF THE INVENTION

The present invention aims at providing drugs having direct angiogenesispromoting action in order to overcome the above-described problems.

The present inventors have extensively studied to find out novel drugshaving direct angiogenesis promoting action. As a result, we have foundthat a piperidine compound represented by the following general formula(1) or salts thereof have direct angiogenesis promoting action as wellas promotion of vascular endothelial cell migration and tube formationeven in an aortic ring culturing assay which was considered not toreflect the vasodilating action. The present invention has beencompleted according to such knowledge.

1. The present invention provides a pharmaceutical composition forpromoting angiogenesis, comprising at least one compound selected from agroup consisting of a piperidine compound represented by the generalformula (1) or salts thereof:

wherein R represents a benzoyl group which may have, as substituents onthe phenyl ring, 1 to 3 groups selected from a group consisting of anamino group, which may have a lower alkanoyl group, and a lower alkylgroup; R¹ represents a hydrogen atom or a lower alkyl group; and R²represents a phenyl lower alkyl group.2. The present invention provides a pharmaceutical composition forpromoting angiogenesis, as is mentioned above, wherein a piperidinecompound is4-[N-methyl-N-(2-phenylethyl)amino]-1-(3,5-dimethyl-4-propionylaminobenzoyl)piperidine.3. The present invention provides a pharmaceutical composition forpromoting angiogenesis, as is mentioned above, wherein thepharmaceutical composition for promoting angiogenesis is a preventive ortherapeutic drug for diseases with insufficient development andregeneration of blood vessels, and various diseases caused by ischemia.4. The present invention provides a pharmaceutical composition forpromoting angiogenesis, as is mentioned above, wherein the diseases withinsufficient development and regeneration of blood vessels, and variousdiseases caused by ischemia are myocardial infarction, angina pectoris,cerebral infarction, senile dementia and various organ damagesaccompanied by diabetes mellitus.

A piperidine compound of the general formula (1) or salts thereofcontained in a pharmaceutical composition for promoting angiogenesis ofthe present invention is a known compound such as described inJP-A-6-340627 (1994).

In the present invention, each group shown in the general formula (1)hereinabove will be described more specifically as follows.

As a lower alkyl group, examples thereof include a linear or branchedalkyl group having 1 to 6 carbon atoms, such as methyl group, ethylgroup, n-propyl group, isopropyl group, n-butyl group, isobutyl group,tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group,n-hexyl group, isohexyl group, 3-methylpentyl group, etc.

As an amino group which may have a lower alkanoyl group, examplesthereof include an amino group, which may have a linear or branchedalkanoyl group having 1 to 6 carbon atoms, such as amino group,formylamino group, acetylamino group, propionylamino group, butyrylaminogroup, isobutyrylamino group, pentanoylamino group,tert-butylcarbonylamino group, hexanoylamino group, etc.

As a benzoyl group which may have 1 to 3 substituents on the phenylring, selected from a group consisting of an amino group which may havea lower alkanoyl group, and a lower alkyl group, examples thereofinclude a benzoyl group, which may have 1 to 3 substituents on thephenyl ring, selected from a group consisting of an amino group whichmay have a linear or branched alkanoyl group having 1 to 6 carbon atoms,and a linear or branched alkyl group having 1 to 6 carbon atoms, such asbenzoyl group, 2-methylbenzoyl group, 3-ethylbenzoyl group,4-n-propylbenzoyl group, 2-isopropylbenzoyl group, 3-n-butylbenzoylgroup, 4-isobutylbenzoyl group, 2-tert-butylbenzoyl group,3-sec-butylbenzoyl group, 4-n-pentylbenzoyl group, 2-neopentylbenzoylgroup, 3-n-hexylbenzoyl group, 4-isohexylbenzoyl group,2-(3-methylpentyl)benzoyl group, 2,3-dimethylbenzoyl group,2,4,6-trimethylbenzoyl group, 2-aminobenzoyl group, 3-formylaminobenzoylgroup, 4-acetylaminobenzoyl group, 2-propionylaminobenzoyl group,3-butyrylaminobenzoyl group, 4-isobutyrylaminobenzoyl group,2-pentanoylaminobenzoyl group, 3-tert-butylcarbonylaminobenzoyl group,4-hexanoylaminobenzoyl group, 2,4-diacetylaminobenzoyl group,2,3,4-triacetylaminobenzoyl group, 3,5-dimethyl-4-propionylaminobenzoylgroup, etc.

As a phenyl lower alkyl group, examples thereof include a phenyl alkylgroup whose alkyl moiety is a linear or branched alkyl group having 1 to6 carbon atoms, such as benzyl group, 1-phenylethyl group, 2-phenylethylgroup, 3-phenylpropyl group, 4-phenylbutyl group, 5-phenylpentyl group,6-phenylhexyl group, 1,1-dimethyl-2-phenylethyl group,2-methyl-3-phenylpropyl group, etc.

Among a piperidine compound represented by the general formula (1) ofthe present invention, a compound having a basic group can easily form asalt with a general pharmacologically acceptable acid. As such an acidincludes, for example, an inorganic acid such as sulfuric acid, nitricacid, hydrochloric acid, phosphoric acid, hydrobromic acid, etc.; and anorganic acid such as acetic acid, p-toluene sulfonic acid, ethanesulfonic acid, oxalic acid, maleic acid, fumaric aid, malic acid,tartaric acid, citric acid, succinic acid, benzoic acid, etc.

Among a piperidine compound represented by the general formula (1) ofthe present invention, a compound having an acidic group can easily forma salt by reacting with a pharmaceutically acceptable basic compound. Anexample of such a basic compound includes sodium hydroxide, potassiumhydroxide, calcium hydroxide, sodium carbonate, potassium carbonate,sodium hydrogen carbonate, potassium hydrogen carbonate, etc.

A piperidine compound of the present invention includes an opticalisomer.

A piperidine compound of the general formula (1) or a salt thereof isusually used in a form of the general pharmaceutical preparation. Thepreparation is produced by using commonly used diluents or excipientssuch as fillers, binders, wetting agents, disintegrators, surface activeagents, lubricants, etc. Various forms of pharmaceutical preparationscan be selected depending on therapeutic objectives, and representativeexamples include tablets, pills, powders, liquids and solutions,suspensions, emulsions, granules, capsules, suppositories, injections(liquids, suspensions, etc.), ointments, etc.

For shaping tablet form, such a carrier can be used, for example,excipients such as lactose, saccharose, sodium chloride, glucose, urea,starch, calcium carbonate, kaolin, crystalline cellulose, silicate,etc.; binders such as water, ethanol, propanol, simple syrup, glucosesolution, starch solution, gelatin solution, carboxymethylcellulose,shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone,etc; disintegrators such as dried starch, sodium alginate, agar powder,laminaran powder, sodium hydrogen carbonate, calcium carbonate,polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate,stearic acid monoglyceride, starch, lactose, etc.; disintegrationsuppressants such as saccharose, stearin, cacao butter, hydrogenatedoil, etc.; absorption enhancers such as quaternary ammonium base, sodiumlauryl sulfate, etc.; moisturizing agents such as glycerine, starch,etc.; adsorbents such as starch, lactose, kaolin, bentonite, colloidalsilicate, etc.; lubricants such as refined talc, stearic acid salt,borate powder, polyethylene glycol, etc. Further, tablets can optionallybe prepared in tablet form coated with conventional coating such assugar-coated tablets, gelatin encapsulated tablets, enteric coatedtablets and film coated tablets, or double layered tablets andmultilayered tablets.

For shaping pill form, such a carrier can be used, for example,excipients such as glucose, lactose, starch, cacao butter, hydrogenatedvegetable oil, kaolin, talc, etc.; binders such as gum arabic powder,tragacanth gum powder, gelatin, ethanol, etc.; disintegrators such aslaminaran, agar, etc.

For shaping suppository form, such a carrier can be used, for example,polyethylene glycol, cacao butter, higher alcohols, esters of higheralcohols, gelatin, semisynthetic glyceride, etc.

Capsules can be prepared by filling a compound of the present inventionby mixing with the above exemplified various carriers into hard gelatincapsules or soft capsules according to conventional capsulation means.

For preparing injection, liquids and solutions, emulsions andsuspensions are sterilized, and are preferably isotonic to blood. Forshaping such forms, such a diluent can be used, for example, water,ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearylalcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fattyacid esters, etc.

Sufficient amount of sodium chloride, glucose or glycerol for preparingan isotonic solution can also be admixed in the pharmaceuticalpreparations, and further conventional solubilizing agents, buffers,soothing agents, and the like can also be added.

If necessary, coloring agents, preservatives, aromatics, flavors,sweeteners, and the like and other drugs can also be added in thepharmaceutical preparation.

For shaping paste, cream and gel form, such a diluent can be used, forexample, white petrolatum, paraffin, glycerin, cellulose derivative,polyethylene glycol, silicon, bentonite, etc.

An amount of a compound of the present invention which should becontained in the pharmaceutical preparation of the present invention isnot especially limited and is selected adequately in a broad range,generally at about 1-70% by weight, preferably 1-30% by weight in thepharmaceutical preparation.

A method for administration of the above pharmaceutical preparation isnot especially limited, and is determined depending on variouspreparation forms, patient age, sex and other conditions and diseaseseverity. For example, tablets, pills, liquids and solutions,suspensions, emulsions, granules and capsules are administered orally.Injections are administered intravenously alone or admixed withconventional fluid replacement such as glucose, amino acids, etc., and,if necessary, are administered alone intramuscularly, intracutaneously,subcutaneously or intraperitoneally. Suppositories are administeredintrarectally.

Dosage of the above pharmaceutical preparation is selected depending ondirection for use, patient age, sex and other conditions and diseaseseverity, and an amount of the compound of the general formula (1) as anactive ingredient is generally about 0.01-10 mg/kg/day. The activeingredient is preferably contained at 0.1-200 mg in a unit dosage form.

A pharmaceutical composition for promoting angiogenesis of the presentinvention is useful as a preventive or therapeutic drug for diseaseswith insufficient development and regeneration of blood vessels, andfurther various diseases caused by ischemia such as myocardialinfarction, angina pectoris, cerebral infarction, senile dementia,various organ damage accompanied by diabetes mellitus, etc.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be more clearly explained by referring toExamples of preparations and pharmacological tests as follows.

PREPARATION EXAMPLE 1

4-[N-methyl-N-(2-phenylethyl)amino]-1-(3,5-dimethyl-4-propionylaminobenzoyl)piperidine(5 mg), starch (132 mg), magnesium stearate (18 mg) and lactose (45 mg)were mixed, and tableted by conventional means to produce tabletscontaining the above described amount of components in a tablet.

PREPARATION EXAMPLE 2

Methylparaben (0.18 g), propylparaben (0.02 g), sodium metabisulfite(0.1 g) and sodium chloride (0.9 g) were dissolved in adequate amount ofdistilled water for injection at 80° C. under stirring. Thus obtainedsolution was cooled to 40° C., and4-[N-methyl-N-(2-phenylethyl)amino]-1-(3,5-dimethyl-4-propionylaminobenzoyl)piperidine(500 mg), polyethylene glycol (molecular weight=4000, 0.3 g) andpolyoxyethylene sorbitan monooleate (0.4 g) were gradually dissolvedtherein, then distilled water for injection was added to the solution toprepare final volume (100 ml). The solution was sterilized by beingaseptically filtered with a proper filter paper, and separatelydispensed each 1 ml into an ampule to prepare injections.

In pharmacological tests hereinbelow, 4-[N-methyl-N-(2-phenylethyl)amino]-1-(3,5-dimethyl-4-propionylaminobenzoyl)piperidine (hereinbelowthis compound is designated as “Test Compound A”) is used as a testcompound.

PHARMACOLOGICAL TEST EXAMPLE 1

Angiogenesis Promoting Action of Test Compound A

The test was conducted according to a report by Nicosia R. F. et al. (InVitro Cell Dev Biol 26, 119-128, 1990). Briefly, thoracic aorta wasremoved from rat under ether anesthesia, and aortic rings were obtainedby cross-sectioning at 1 mm intervals. The aortic rings were embeddedinto type I collagen gel (Koken Co.). Further, MCDB131 medium (Gibco BRLCo.) containing the Test Compound A was added to the upper layer, andthe medium was exchanged every three days. After 7 days from theculture, micro-vessels newly grown from a cutting section of the aorticring specimens were stained with alkaline phosphatase, then numbers ofmicro-vessels observed based on digital camera image were counted byusing Scion Image (Scion Corp.). The statistical analysis was performedas follows: Data were analyzed using Dunnett's test with randamizedblock design. The difference between test compound groups andnon-treated group were considered statistical significant at P<0.05.

The results are shown in Table 1. As is shown in Table 1, the TestCompound A indicated significantly increased numbers of newly grownmicro-vessels with dose dependent manner in the thoracic aortic ringspecimens of rat, as compared with those of a non-treated control group.Accordingly, the Test Compound A was clearly demonstrated to haveangiogenesis promoting action.

TABLE 1 Concentration Number of (−Log M) Number micro-vessels Control 620 ± 2 Test 12 7 24 ± 3 Compound A 11 7 22 ± 3 10 7 30 ± 3 * 9 7 27 ± 28 7 32 ± 4 * 7 7 33 ± 3 * 6 6 35 ± 4 * Values were expressed as the mean± SE ** P < 0.05 vs. control

PHARMACOLOGICAL TEST EXAMPLE 2

Effect of Test Compound A on Vascular Endothelial Cell Migration

The test was conducted according to a report by Witzenbichler, B. et al.(J. Biol. Chem., 273, 18514-18521, 1998). Briefly, human aorticendothelial cells (Cambrex Corp.) were cultured in MCDB131 medium toconfluent state. Cell migration was tested by using a 48 well microchemotaxis chamber (Neuro Probe Inc.). The Test Compound A was addedinto the lower layer of a well, while cells, 1×10⁴ cells per well, wereadded into the upper layer, and those were cultured for 4 hours. Cellswere immobilized with methanol and cells in the upper layer of a filterwere removed, and cells migrated on the filter were stained with DiffQuick (International Reagents Corp.), then numbers of migratedendothelial cells observed from digital camera image were counted byusing Scion Image (Scion Corp.). The statistical analysis was performedas follows: Data were analyzed using Williams' test after linearregression analysis. The difference between test compound groups andnon-treated group were considered statistical significant at P<0.05.

The results are shown in Table 2. From the results in Table 2, the TestCompound A indicated significantly increased numbers of migration cellswith dose dependent manner as compared with those of a non-treatedcontrol group, then the Test Compound A was confirmed to promotemigration of vascular endothelial cells.

TABLE 2 Concentration Number of (−Log M) Number Migration cell Control 434 ± 8 Test 12 4 41 ± 8 Compound A 11 4 42 ± 6 10 4 49 ± 6 9 4 64 ± 8 **8 4 64 ± 7 ** Values were expressed as the mean ± SE ** P < 0.01 vs.control

PHARMACOLOGICAL TEST EXAMPLE 3

Effect of Test Compound A on Luminalization of Vascular EndothelialCells

The test was conducted according to a report by Yasunaga, C. et al. (LabInvest, 1989; 61: 698-704). Briefly, human aortic endothelial cells(Cambrex Corp.) were cultured in MCDB131 medium to confluent state.Cells prepared with medium containing the Test Compound A, 5×10⁴cells/well, were added on cell matrix gel (Nitta Gelatin Co.) andcultured for 4 hours to adhere cells. After medium removal, gel wasadded to embed cells, and medium containing the drug was added on theupper layer, then length of the formed tube was measured on the thirdday based on digital camera image by using Scion Image (Scion Corp.).The statistical analysis was performed as follows: Data were analyzedusing Williams' test after linear regression analysis. The differencebetween test compound groups and non-treated group were consideredstatistical significant at P<0.05.

The results are shown in Table 3. From the results in Table 3, the TestCompound A indicated significantly increased length of formed tube withdose dependent manner as compared with those of a non-treated controlgroup, and the test compound A was confirmed to promote tube formationof vascular endothelial cells.

TABLE 3 Concentration Length of formed (−Log M) Number tube (Pixel)Control 4 13.3 ± 0.4 Test 8 4 15.7 ± 0.9 Compound A 7 4 32.2 ± 3.8 ** 64 48.4 ± 5.3 ** Values were expressed as the mean ± SE ** P < 0.01 vs.control

The invention claimed is:
 1. A method for promoting angiogenesis, whichcomprises administering to a person in need of improvement of bloodcirculation a pharmaceutical composition having, as the activeingredient, at least one compound selected from a group consisting of apiperidine compound represented by the formula (1) or salts thereof:

wherein R represents a benzoyl group which may have, as substituents onthe phenyl ring, 1 to 3 groups selected from a group consisting of anamino group, which may have a lower alkanoyl group, and a lower alkylgroup; R¹ represents a hydrogen atom or a lower alkyl group; and R²represents a phenyl lower alkyl group.
 2. The method according to claim1, wherein the piperidine compound is4-[N-methyl-N-(2-phenylethyl)amino]-1-(3,5-dimethyl-4-propionylaminobenzoyl)piperidine.3. The method according to claim 1 or claim 2, wherein thepharmaceutical composition is a therapeutic drug for diseases withinsufficient development and regeneration of blood vessels, and diseasescaused by ischemia.
 4. The method according to claim 3, wherein thediseases with insufficient development and regeneration of bloodvessels, and diseases caused by ischemia are selected from the groupconsisting of myocardial infarction, angina pectoris, cerebralinfarction, and senile dementia.